The derivatives of phosphoric acid have been shown to have a wide range of biological utility (Emsley and Hall (1976) in The Chemistry of phosphorus: Chapter 12 Biophosphorus Chemistry pp. 471–510 Harper and Row: London, England). In turn, molecules that mimic phosphoric acid and its derivatives have been shown to work as biological effectors and are often used as diagnostic and therapeutic agents (Uhlmann and Peyman (1990) Chem. Rev. 90: 544). Examples of these derivatives are phosphonocarboxylates (Becker et al. (1977) Antimicrob. Agents Chemother. 11: 919), phosphorothioates (Eckstein (1989) Trends Biochem. Sci. 14: 97), phosphorodithioates (Nielsen et al. (1988) Tetrahedron Lett. 29: 2911), methylphosphonates (Miller and Ts'o (1988) Annu. Rep. Med. Chem. 23: 295), and phosphoramidates (Iyer et al. (1996) Tetrahedron Lett. 37: 1543).
Phosphonocarboxylate mimics of phosphoric acid, specifically phosphonoformic acid and phosphonoacetic acid, have been shown to be especially useful as biological effectors and have been used as therapeutic agents (Shipkowitz et al. (1973) Appl. Microbiol. 26: 264; Helgestrand et al. (1978) Science 201: 819). The syntheses of phosphonoformic acid (Nylen (1924) Chem Berichte. 57: 1023) and phosphonoacetic acid (Basinger et al. (1959) J. Org. Chem. 24: 434) have relied upon the introduction of the carboxylate group onto the phosphorus moiety through an oxidative transformation such as a Michaelis-Arbuzov reaction (Arbuzov and Dunin (1914) J. Chem. Soc. 653; Arbuzov (1964) Pure Appl. Chem. 9: 307). The resulting phosphonocarboxylic acid products are in the oxidation state P(V). Once the phosphorus atom is in this pentacoordinate oxidation state the products are typically very stable. However, these stable products are difficult and sometimes impossible to utilize in performing high yielding chemical transformations, chemical couplings, or chemical derivatizations. As a result of the low chemical reactivity of these pentacoordinate phosphorus molecules, many biologically important molecules that exist as phosphoric acid derivatives have not been mimicked with phosphonocarboxylic acid derivatives (Hildebrand (1983), in The Role of Phosphonates in Living Systems: Chapters 5 & 6, pp. 97–169, CRC Press Inc: Boca Raton, USA).
Two clear examples of biologically important molecules that exist naturally as phosphoric acid derivatives and have not been mimicked as phosphonocarboxylic acid derivatives are the polynucleotides DNA and RNA. Polynucleotides modified at the phosphodiester internucleotide linkage are of significant interest to the emerging fields of antisense therapeutics, nucleic acid diagnostics, and genomics. Phosphorus-containing chemical compounds and compositions that have been successfully utilized to enable the synthesis of polynucleotides have been frequently reviewed in the scientific literature (Verma et al. (1998) Annu. Rev. Biochem. 67:99; Sekine et. al. (1998) Nucleosides and Nucleotides 17:2033; Iyer et al (1999) Curr. Opin. Mol. Ther. 1:344). The successful chemical synthesis of polynucleotides or modified polynucleotides is a task especially dependent upon the ability to find and employ phosphorus-containing compounds that enable high yield chemical couplings and chemical transformations (Caruthers (1985) Science 230:281; Caruthers et al., U.S. Pat. No. 4,415,732, issued Nov. 15, 1983). To enable the chemical synthesis of polynucleotides or modified polynucleotides, the phosphorus compounds used must be able to perform high yield coupling reactions that are general to the four nucleobases and specific for the desired polynucleotide products. High yield coupling efficiencies for the formation of internucleotide bonds are necessary in order to enable the synthesis of biologically relevant lengths of polynucleotides (Koster et al., U.S. Pat. No. 4,725,677 issued Feb. 16, 1988), wherein a “biologically relevant length” is a length that allows the polynucleotide to stably and specifically bind to other polynucleotides by hybridization through base-pairing interactions. Stable binding of polynucleotides to other polynucleotides via hybridization is also affected by temperature, salt concentration, nucleotide sequence, and other factors, as has been extensively discussed in the literature; see, e.g., Sanger (1984) in Principles of Nucleic Acid Structure: Chapter 6, pp. 116–158 (Springer-Verlag: New York, USA).
The need for high yield coupling reactions in synthesizing polynucleotides of a biologically relevant length is due to the mathematical relationship between the final yield of the desired polynucleotide product and the efficiency for each individual coupling reaction giving rise to a new internucleotide bond. The final yield of the desired polynucleotide product is a multiplication product of all individual coupling and deprotection steps required in achieving that product. As a result, the yield of the final polynucleotide product decreases exponentially with a linear decrease in the coupling efficiency. That is, the effect of the coupling efficiency on the overall yield of product can be described by the equation Y=XN, where Y is the fractional overall yield, X is the fractional coupling efficiency, and N is the number of couplings. For the synthesis of a typical polynucleotide 20 nucleotides in length with 19 internucleotide linkages, 19 coupling reactions are involved and the overall yield is given by Y=X19. The table below illustrates the relationship between the coupling efficiency (X) and overall yield of polynucleotide product (Y).
X0.100.200.300.400.500.600.700.800.900.950.99Y1−195−141−103−32−66−51−31−20.140.380.82
As clearly illustrated by this example, and as well known by those skilled in the art, the synthesis of a 20-mer polynucleotide is not possible until the coupling efficiency achieved during synthesis approaches 90% or greater. Only at these coupling efficiencies can full-length polynucleotides be reproducibly isolated and purified from the reaction mixtures. As a further example for illustration, at an 80% coupling efficiency, the theoretical maximum amount of full-length product (Y), after 19 couplings, is 1.4%. However, the overall yield shown in the table above is a simplification that considers only the effect of the efficiency for the formation of the internucleotide bond. The actual overall yield of a polynucleotide product is additionally adversely affected by any inefficiency in deprotection reactions used during synthesis, post-synthesis, or from side-reactions leading to undesired products. The ability to isolate a full-length polynucleotide product, 20 nucleotides in length, from a polynucleotide synthesis that achieves an 80% per cycle coupling efficiency is precarious and rarely reproducible, and directly linked to the yield of the individual deprotection reactions following each coupling step. As a direct result of these requirements for high yield reactions, the chemical synthesis of polynucleotides has been accomplished by only very few methods; see Brown (1983) in Protocols for Oligonucleotides and Analogs: Chapters 1, pp. 1–17 (Humana Press: Totowa, N.J., USA, Ed. S. Agrawal). Each of the methods that has enabled the chemical synthesis of polynucleotides has in turn been enabled by the development of high yielding coupling reactions at the phosphorus moiety, in concert with the development of high yielding deprotection reactions. The fact that so few methods have enabled polynucleotide synthesis is a direct result of the difficulty of performing a long series of sequential chemical reactions in quantitative or near-quantitative yields.
The chemical synthesis of polynucleotides containing the naturally occurring phosphodiester linkage was originally accomplished using nucleotide building blocks known as “diester intermediates.” These “diester” building blocks were nucleotide monomers on which the heterocyclic bases and exposed hydroxyl functionality were chemically protected by blocking groups, and the pendant phosphate group activated for transesterification reactions by the formation of a “phosphodiester” (Gilham & Khorana (1958) J. Amer. Chem Soc. 80: 6212). The low reactivity of these activated P(V) monomers resulted in low synthetic yields for internucleotide bond formation. The typical yield for the coupling of nucleoside phosphodiester monomers to nucleoside or nucleotide hydroxyl groups is in the range of 20–50%. The low synthetic yields achieved by these chemical coupling reactions limited this method to the synthesis of monomer, dimer, and trimer products. The synthesis of biologically relevant lengths of polynucleotides from these monomer, dimer, and trimer blocks was accomplished by the use of an enzymatic ligation reaction (Khorana (1966) The Harvey Lectures, ser.62, pp. 79–106; Khorana, H. G. (1979) Science 203: 614). This enzymatic process was dependent upon these monomer, dimer, and trimer blocks acting as high yielding substrates for enzymatic ligation reactions, and on the enzymatic reactions producing the desired naturally occurring phosphodiester internucleotide bond. The complete chemical synthesis of these polynucleotides could not be demonstrated as a result of the low coupling efficiencies of phosphodiester intermediates for the formation of internucleotide bonds.
The complete chemical synthesis of polynucleotides containing naturally occurring phosphodiester internucleotide linkages was first made possible by the development of phosphotriester reactive intermediates (Narang et. al. (1980) Methods in Enzymology 65:610) and phosphite reactive intermediates (Letsinger & Lunsford (1976) J. Am. Chem. Soc. 98:3655; Beaucage & Caruthers (1981) Tetrahedron Lett. 22:1859). Phosphotriester reactive intermediates produced coupling efficiencies for the formation of internucleotide bonds in the range of 65–87% depending upon the sequence and condensing agent utilized (Efimov et. al. (1982) Nucleic Acids Research 10:6675). In the lower end of this range of coupling efficiencies, the enzymatic ligation of block-coupled products was required to enable the synthesis and isolation of polynucleotides. In the upper end of the range of coupling efficiencies, it was difficult but possible to isolate full-length polynucleotides from the reaction products of a complete chemical synthesis. The subsequent development of phosphite reactive intermediates produced coupling efficiencies for the formation of internucleotide bonds in the range of 90–99%. It was the invention of these phosphite chemical compositions of protected nucleosides, in concert with effective protecting group chemistry, that enabled the routine chemical synthesis of native polynucleotides (Matteucci & Caruthers (1981) J. Am. Chem. Soc. 103: 3185).
The chemical synthesis of phosphorus-backbone modified polynucleotides is more difficult than the chemical synthesis of naturally occurring phosphodiester-linked polynucleotides. Modification of the phosphorus backbone, in most cases, precludes the use of high yielding enzymatic ligation methods. The enzymes that are typically used for the formation of internucleotide bonds have substrate fidelity requirements that make it difficult if not impossible to use them to form backbone-modified internucleotide bonds. A few phosphorus backbone modifications, such as phosphorothioates, have been formed using enzymatic incorporation of modified nucleotides or enzymatic ligation reactions, but these modifications tend to be rare and the reactions much less efficient than the formation of native internucleotide bonds (Eckstein (1985) Annu. Rev. Biochem. 54:367). Without the ability to overcome low coupling efficiencies using enzymatic ligation of dimer and trimer blocks, the enablement of backbone-modified polynucleotides is even more rigorously tied to the need for high yielding coupling reactions and high yielding deprotection reactions than is the synthesis of phosphodiester-linked polynucleotides.
Nucleotide monomers of phosphonocarboxylates, phosphonoformic acid and phosphonoacetic acid (Heimer et. al., U.S. Pat. No. 4,056,673 issued Nov. 1, 1977; Sekine et. al. (1982) Bull. Chem. Soc. Jpn. 55: 239; Griengl et. al. (1988) J. Med. Chem. 31: 1831; Lambert et. al. (1989) J. Med. Chem. 32: 367) have been prepared using protected nucleosides and the activated transesterification techniques developed for phosphodiester and phosphotriester coupling of internucleotide bonds (Shaller et. al. (1963) J. Am. Chem. Soc. 85: 3821; Amarnath et al. (1977) Chem. Rev. 77: 183). These modified nucleotide monomers have been shown to be inhibitory to DNA and RNA polymerases and have not proved to be efficient substrates for enzymatic incorporation or ligation. As a result, the enablement of phosphonocarboxylate derivatives of polynucleotides requires the ability to perform complete chemical synthesis, in turn requiring high yielding coupling reactions and high yielding deprotection reactions.
Prior to the current invention, attempts by the present inventor to chemically synthesize phosphonocarboxylate modified polynucleotides were performed using the chemical reactions previously reported for coupling alkyl esters of phosphonoformic acid and phosphonoacetic acid to protected nucleosides. 3′-Phosphonocarboxylate protected nucleotide monomers were prepared and isolated by literature protocols. A series of attempts were then made to produce polynucleotides with phosphonocarboxylate-modified internucleotide linkages by applying one of the many standard phosphotriester condensing agents on solid-phase (Stawinski et. al. (1977) Nucleic Acids Res. 4: 353; Reese et. al. (1978) Tetrahedron Lett. 19: 2727). All of these attempts to form internucleotide bonds using activated transesterification methods on the P(V) phosphonocarboxylates of protected nucleosides, gave coupling efficiencies too poor to enable the synthesis of polynucleotides. Although the coupling efficiencies were extremely low (<10%), solution-phase coupling of these modified nucleotides to 3′-protected nucleosides allowed for isolation of a small amount of thymidine-thymidylate protected dimers with phosphonoformate and phosphonoacetate internucleotide bonds. Attempts to deprotect the ethyl or methyl esters of the carboxylic acid phosphonate dimers, by the hydrolysis methods previously published for nucleotide monophosphonocarboxylates, led to cleavage of the internucleotide bond. Using base-catalyzed, nucleophilic hydrolysis conditions to deprotect the carboxylic acid methyl or ethyl esters of phosphonoformate and phosphonoacetate thymidine-thymidylate dimers, resulted in significant cleavage of the phosphorus-carbon bond (40%–100%). Analysis of the products from these nucleophilic hydrolysis reactions demonstrated cleavage of the phosphorus-carbon bond that in turn resulted in both cleavage of the internucleotide bond, and conversion of the modified internucleotide bond to a phosphodiester bond. A subsequent literature study confirmed these observations and revealed that the phosphorus-carbon bond of acylphosphonates was susceptible to cleavage under the conditions of carboxylate ester hydrolysis. The facile loss of similar phosphorus-carbon bonds had been previously observed under the conditions of nucleophilic hydrolysis and a mechanism proposed (Sekine et al. (1980) J. Org. Chem. 45: 4162; Narayanan et. al (1979) J. Am. Chem. Soc. 101: 109; Kluger et. al. (1978) J. Am. Chem. Soc. 100: 7382). The nucleophilic attack of hydroxide or other nucleophile on the carbonyl of phosphonocarboxylates result in the formation of a tetrahedral intermediate containing a localized negative charge on the oxygen of the carbonyl. Further rearrangement of this intermediate results in reaction products that favor cleavage of the phosphorus-carbon bond rather than the desired substitution by hydroxide. Once again, there is a high yield requirement for these deprotection reactions in order to enable the synthesis and isolation of phosphonocarboxylic acid modified polynucleotides. Non-quantitative deprotection reactions at the internucleotide linkage or rearrangement products that lead to undesired side-products has an exponentially negative effect on the yield of full-length polynucleotide product. Moderate to low yields for the removal of protecting groups, or cleavage of the phosphorus-carbon bond during deprotection of the carboxylic acid thus directly prevents the enablement of these modified polynucleotides. Although one reference describes synthesis of phosphonocarboxylic acid modified polynucleotides as feasible (Cook et al., International Patent Publication No. WO 93/10140), in fact the methods described, or similar activated transesterification methods, do not enable the synthesis of phosphonocarboxylic acid modified polynucleotides.
Rudolph et. al (1996) Nucleosides and Nucleotides 15:1725 describe the solution-phase synthesis of thymidine-thymidylate dimers containing methyl phosphonoacetate internucleotide bonds using an activated transesterification method with 1-(2-mesitylene-sulfonyl)-3-nitro-1,2,4-triazole (MSNT) and a pentavalent phosphonoacetate derivative. In order to incorporate this dimer modification into longer polymers the authors derivatized the dimer with a standard phosphoramidite reagent (Atkinson et al. (1984) Oligonucleotide Synthesis: A Practical Approach, Gait, Ed., IRL Press, Oxford, pp 41–45) and used the high yielding formation of native internucleotide phosphodiester bonds to assemble longer polymers, containing only the nucleobase thymidine, with the assumption that every other linkage in the resulting reaction mixture would be a phosphonoacetate. The publication additionally reported that the phosphonoacetate group is labile to ammonium hydroxide, hydrazine, ethylenediamine, triethylamine/water, and piperidine/water, resulting in the cleavage of the internucleotide bond. The conditions reported to give complete cleavage of the internucleotide bond are the same conditions that are reported in WO 93/10140. As in the aforementioned PCT publication, Rudolph et al. clearly demonstrated that the activated transesterification coupling of protected nucleoside, P(V) alkyl phosphonocarboxylates and subsequent hydrolytic cleavage of alkyl ester protected carboxylic acid groups does not enable the synthesis of phosphonocarboxylic acid modified polynucleotides.
There are few reported phosphorus chemical compositions in the oxidation state P(III) having protected carboxylic acid functional groups. Reports of phosphinylacetic acid derivatives are uncommon, and have been exclusively studied for their unique physical (Matrosov et. al. (1972) Zh. Obshch. Khim. 42: 1695) and chemical (Podlahova, J. (1978) Collection Czechoslov. Chem. Commun. 43: 57) properties rather than their use as chemical synthons. Also see Novikova et. al. (1976) Zh. Obshch. Khim. 46: 575; Novikova et. al. (1976) Zh. Obshch. Khim. 46: 2213; and Stepanov et. al. (1979) Zh. Obshch. Khim. 49: 2389)
The low chemical reactivity of pentacoordinate phosphonocarboxylate molecules has prevented the incorporation of internucleotide phosphonocarboxylate moieties into many biologically important molecules. Preparation of oligonucleotides containing internucleotide phosphonocarboxylate moieties would require chemical compositions of phosphorus that perform high yielding chemical couplings and chemical transformations. More particularly, chemical synthesis of phosphonocarboxylate oligonucleotides and polynucleotides (DNA and RNA) would require both high yielding coupling reactions at the phosphorus moiety and high yielding deprotection reactions at the carboxylate moiety, with the phosphorus-carboxylate moiety left intact.